A preponderance of evidence has identified nontuberculous mycobacteria (NTM) as important pathogens in cystic fibrosis (CF) and other obstructive lung diseases. Currently, airway cultures are the ?gold standard? by which all diagnosis and treatment decisions are based. The lack of sensitive and specific markers of NTM in the airway impedes patient care and clinical trial design. Culture-independent markers of NTM infection have the potential to overcome many of the limitations of standard NTM cultures, especially the very slow growth (up to 8 weeks), inability to quantitate bacterial burden, and low sensitivity due to required decontamination procedures. Preliminary data from analysis of volatile metabolites and multiplex gene sequencing performed simultaneously on the same sputum samples have demonstrated rapid, quantifiable detection of the presence of NTM that exceeds the sensitivity of standard cultures. In 2018, the CF Foundation-sponsored PREDICT (PRospective Evaluation of NTM Disease In CysTic Fibrosis, NCT02073409) and PATIENCE (Prospective Algorithm for Treatment of NTM in Cystic Fibrosis, NCT02419989) trials will be expanded to 10 CF Programs nationwide to standardize the diagnosis and test a protocol-based approach to treatment of NTM pulmonary disease. These trials represent an ideal platform to advance development of molecular markers of NTM disease status. In response to PAR-18-643 NHLBI Clinical Ancillary Studies, this proposal will test the Central Hypothesis: Culture-independent marker sets have advantages over sputum cultures for detecting presence of bacteria, predicting clinical significance and assessing treatment response of NTM in the CF airway. As a time-sensitive ancillary study to the PREDICT and PATIENCE trials, Aim 1 will test the utility of sputum volatile metabolites as culture-independent markers of airway NTM. Aim 2 will test the utility of sputum multiplex gene sequencing as molecular markers of airway NTM, and Aim 3 will test the combination of volatile metabolites and multiplex gene sequencing data combined with standard culture results to identify subjects more likely to develop NTM disease and/or respond to treatment. If successful, the cross-sectional and longitudinal analyses of samples from pediatric and adult patients with well-defined NTM infection status will reveal the strengths and weakness of each assay alone and combined, in comparison to standard culture for the detection and identification of NTM in the presence of bacterial co- infection. These markers can be applied to periodic screening of patient populations at risk for NTM to facilitate early detection. In addition, these assays have the potential to differentiate between indolent infection and NTM disease, as well as longitudinal monitoring of response to treatment and determination if eradication of the pathogen has been achieved or a subclinical threshold has been reached, which could signal end of treatment. The long-term goal is to deliver validated markers of NTM airway disease to the CF patient population, with applicability to all patient populations with respiratory conditions at risk for NTM disease.